Abstract
Abstract: :
Purpose: The Cngb1 locus is complex encoding the cGMP–gated cation channel ß–subunit (cngb) and related GARP proteins in the retina and alternatively spliced isoforms in several non–retinal tissues. We created a Cngb1 knockout mouse to determine the function of the proteins expressed in the retina. Methods: Knockout (KO) mice were created using ES cell technology to target deletion of the predicted Cngb1 promoter region and exons 1 and 2. Western blot assays were performed on purified ROS homogenates using antibodies against several photoreceptor proteins. Retinal sections were examined by light microscopy. Scotopic ERG analysis was performed on KO and wt mice to assess visual function. Results: Molecular analysis confirmed the absence of the targeted region of the Cngb1 locus. Antibodies against a common N–terminal epitope in GARP–1, GARP–2, cngb, and an anti–cnga antibody failed to recognize any protein in KO mouse ROS protein homogenates. Antibodies against ABCR, Rom1, rhodopsin, and peripherin–2, however, showed similar amounts of product in +/+, –/+ and –/– samples. KO mice show a progressive retinal degeneration that primarily affects the photoreceptors. At 30 days the outer segments appear disorganized and slightly reduced in length. By three months the ONL was reduced to 5 rows and the outer segments are disorganized and sparse in number with significant cell debris apparent. The OPL is compromised by invading ONL and INL cell bodies. In the KO mouse b–wave sensitivity was dramatically reduced (20 to 50 fold), but maximum amplitude was maintained up to 100 days. Older animals showed reduced b–wave amplitudes as well as decreased sensitivity. Conclusions: The absence of the channel α–subunit from ROS membranes indicates that the ß–subunit is required for targeting of the α–subunit to the membrane in contrast to results of in vitro studies of heterologously expressed channel subunits. At least one of the three major retinal proteins encoded by the Cngb1 locus is essential to maintain the structural integrity and visual function of the rod photoreceptor.
Keywords: retinal degenerations: cell biology • retina: distal (photoreceptors, horizontal cells, bipolar cells) • signal transduction